Development of Adiponectin Receptor C-Terminal Fragment Bioassays

Abstract

The presence of adiponectin receptor C terminal fragment (AdipoR CTF) in human blood was discovered using immune-affinity mass spectroscopy techniques (Pugia et al., Clin Proteomics 5:156–162, 2009). Deficiency in diabetic patients was observed and followed up by enzyme linked immunoabsorbent assays (ELISA) with generation of a set of antibodies for AdipoR1 and R2 CTF. Additional western blot analysis identified the majority of AdipoR CTF in human, rat and rabbit blood was covalently bound to immunoglobulins (Ig-CTF) as the primary native form. Bound CTF in patient plasma was found at 95–4900 ng/mL and to be <0.2 % of all plasma IgG. Free CTF was found at 0.05–5.0 ng/mL. CTF was shown to be covalently bound to immunoglobulin and only released upon basic conditions and not simply by breaking the disulfide bonds. Mass spectroscopy peptide enrichment assays utilizing trypsin digests were unable to digest bound CTF and basic treatment was required to liberate free form. Antibodies for AdipoR1 and R2 CTF were paired in sandwich ELISA suitable for measurement Ig-CTF bound forms for clinical patient testing and free CTF forms for animal model testing. Analytical validation demonstrated <10 % between sample and <10 % within patient reproducibility. Calibration, precision, and stability were acceptable. No cross reactivity or interference was observed. Simple sample preparation was demonstrated as a benefit over mass spectroscopic peptide enrichment assays analysis.

Notes

Acknowledgements

We would like to acknowledge the work done by Siemens Healthcare Diagnostic people including Mary Foltz, Jill Perry, David Brock, Qingping Jiang, Yu Hui Lin, Jim Freeman and others who helped prepare antibody, conjugates, peptides, and assay formats. We would like to acknowledge the mass spectroscopy work done by the SELDI group of Dr. Saeed A. Jortani, Department of Pathology and Laboratory Medicine, University of Louisville, School of Medicine, by the SISCAPA group of Dr. Terry W. Pearson and Dr. Matthew Pope of SISCAPA Technologies at the University of Victoria BC Canada Victoria., and by the i-MALDI group of Dr. Christoph Borchers & Dr Andrew Munk of MRM Proteomics at the University of Victoria BC Canada Victoria.

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